EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT

Transcription

1 EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT J. E. Adskveg, H. Förster, D. Thompson, D. Felts, D. Cry, K. Nguyen, J. Connell, R. Buchner Cooperting: L. Wde, Aryst LifeScience ABSTRACT Our reserch efforts include supporting the full nd tenttively pproved 2013 emergency registrtion of copper-mncoze comintions s the most consistent nd proven effective tretment for mnging wlnut light cused y copper-resistnt popultions of the cteril pthogen Xnthomons roricol pv. juglndis. We provided specific informtion s requested y the US-EPA on: 1) the distriution of copper-resistnce in the stte; 2) inclusion of dditionl counties for the emergency registrtion; 3) sustntition of continued efficcy nd demonstrle crop yield improvement of copper-mncoze pplictions; nd 4) evlutions of new lterntive tretments to copper for disese mngement. Although disese ws high in some loctions in 2012 nd the occurrence of copper resistnce widespred from the centrl to northern wlnut production res, mncoze (UPI Mnzte) nd copper (severl new formultions such s Kocide 3000, Bdge X2, Cuprofix, nd Chmp 30)-mncoze comintions were modertely nd highly inhiitory, respectively, to ll 296 strins collected from 39 orchrds in 10 counties. In 2012, ksugmycin (Ksumin 2L) in comintions with copper or mncoze ws highly effective nd ws gin the most promising new tretment towrds developing n integrted mngement progrm tht will reduce totl use of ny one product in the environment nd reduce the risk for selecting resistnce in pthogen popultions. Ksugmycin is currently pending US-EPA registrtion in Mrch Additionlly, we continued to evlute chemicl, iologicl, nturl, nd systemic cquired resistnce (SAR) compounds s potentil future lterntive nd rottionl tretments. Formultions nd rtes of Regli continued to e evluted in efforts to improve performnce nd consistency. Moleculr studies on pthogen diversity re ongoing nd demonstrte tht the pthogen hs limited diversity within orchrds ut high diversity mong orchrds cross the stte. In our reserch on ud popultions of the pthogen, we found tht wlnut uds support diverse numer of cteril species with t lest eight different gener producing yellow colonies tht co-exist with the pthogen. These cteri cnnot e esily distinguished during isoltion nd require culturl chrcteriztion using selective medi nd moleculr methods. Disese incidence during the seson did not correlte with popultions of the pthogen in uds collected in the fll. Thus, strtegy of using smll smples of 30 to 50 uds per orchrd nd culturing cteril popultions on selective medi is not dvised t this time for predicting susequent spring disese levels nd mngement prctices. Becuse reltively low inoculum levels of the pthogen in uds cn cuse primry fruit infections nd rpidly increse pthogen popultions under fvorle environmentl conditions, high potentil for n epidemic exists even when ud popultions re t low to moderte levels. In our strtegy, we re evluting disese levels in orchrds in erly summer to identify high-risk sites. Additionlly, we re using wether forecsts in the susequent spring (fter the pistillteflower-emergence ppliction of ctericide) s the most effective wy to optimize dditionl tretment timings for mnging the disese. Cliforni Wlnut Bord 309 Wlnut Reserch Reports 2012

2 OBJECTIVES I. New tretments - Evlute the toxicity of lterntive copper (i.e., the reduced-mce products such s Kocide 3000 nd Bdge X2), non-copper sed chemicls (e.g., Ksumin, EBDC compounds nd other fungicides), iologicls (e.g., Actinovte, Blossom Protect), nd other experimentl mterils such s residul oxidizers nd novel snitizing gents ginst X. roricol pv. juglndis nd evlute the efficcy of these mterils for mnging wlnut light. A) Comprtive efficcy of new copper ctericides such s new formultions in field trils under mient nd simulted rin systems t the Kerney Agriculturl Reserch nd Extension (KARE) Center, UC Dvis, nd in commercil orchrds in Butte or Sutter Co. Support the full-registrtion of mncoze in copper mixtures for the mngement of wlnut light Evlute copper persistence with nd without djuvnts under simulted rin B) Optimize the performnce of ksugmycin formultions (e.g., Ksumin 2L, 8L, 10L) in mixture rottions with copper, EBDC compounds (e.g., Mnzte, Dithne) nd other fungicides (e.g., cptn, Quintec, polyoxin-d). Comintions of the ntiiotic with copper, EBDC, nd selected register-le fungicides for sustined usge nd stewrdship of the ntiiotic C) Investigte the use of novel chemistries for snitizing trees (e.g., AN-77V2) tht utilize residul oxidizers to protect trees for extended periods nd phosphonte pesticides with reported ctivity ginst cteril diseses. Evlute these products for use in comintion with iologicls (e.g., Actinovte, Blossom Protect) nd inducers of host defense mechnisms (e.g., Citrox, ProAlexin, BioForge) or nti-trnspirnt inducers (e.g., Root- Zone, CoMo Clssic) s tretments for mnging wlnut light. II. Epidemiology - Continue to evlute disese development throughout the spring nd monitor environmentl prmeters (e.g., lef wetness, precipittion, temperture, nd reltive humidity) tht re conducive to cteril infection of wlnut tissues nd evlute popultions of the cteril pthogen using moleculr pproches A) Develop nd evlute dew point nd precipittion-temperture-sed version of XnthoCst on University wesites (ongoing) in coopertion with UC-IPM nd CIMIS progrms for utilizing temperture nd precipittion, dew point temperture, or reltive humidity dt insted of lef wetness for the model. B) Evlute spring timings efore nd fter mjor rinfll or simulted rin events nd set upper temperture limits for ctericide pplictions (e.g., >27C). C) Evlute diversity within pthogen popultions nd rpid identifiction of the pthogen using moleculr methods. III. Nturl host resistnce of new wlnut genotypes ginst wlnut light. New genotypes will e evluted including Forde, Ivnhoe, nd others t Kerney Ag Center. SIGNIFICANT FINDINGS Incidence of wlnut light t mny loctions ws very high (>50%) in 2012, including in orchrds where severl copper-mnzte tretments were pplied. To find possile reson for the filure of ctericide tretments, 296 isoltes of Xnthomons roricol pv. juglndis were collected from 39 orchrd loctions in 10 counties. Copper resistnce ws found in ll ut seven Cliforni Wlnut Bord 310 Wlnut Reserch Reports 2012

3 orchrds nd ll isoltes were sensitive to mncoze. This led to the conclusion tht coppermncoze tretments did not fil, ut high inoculum levels, poor timing, reduced numer of pplictions (<4), nd more fvorle environmentl conditions in 2011 nd 2012 were mostly responsile for the disese outrek. Our ctericide efficcy trils on wlnut light in 2012 focused on the comprtive effectiveness of copper products (Bdge X2, Kocide 3000, Cuprofix, Chmp 30), the ntiiotic Ksumin, iologicls (Regli, Citrox, Actinovte, Blossom Protect), tretments tht ffect plnt growth or physiology (Actigrd, ProAlexin, CoMo-Clssic, RootZone), the fungicides Quintec nd Cptn, nd dditionl new tretments (phosphontes, polyoxin-d, AN-77V2), s well s selected mixtures. Results were otined in six field trils, including three trils t commercil sites. The results cn e summrized s follows: 1. Copper-mncoze tretments were effective in ll trils conducted, except t one commercil site where ll tretments, including grower-pplied tretments, were less effective in mnging the disese. Copper tretments (Kocide 3000, Bdge X2, Cuprofix, nd Chmp 30) when mixed with Mnzte were sttisticlly similr in their effectiveness. Numericlly, Bdge X2 resulted in the lowest incidence of disese (8.3% s compred to 41.8% in the check). 2. Tretments with Ksumin y itself were very effective under low disese pressure. Under high disese pressure (31.3% or 41.8% disese in the control), Ksumin-Mnzte ws sttisticlly similrly effective to copper-mnzte tretments. 3. In n orchrd with copper-sensitive pthogen popultions, Regli (20%.i. experimentl formultion) ws only effective when pplied t rtes of 64 fl. oz. or higher. Phytotoxicity ws oserved t the high-rte usge. The mximum rte of the commercil formultion of Regli (5%.i.) is 128 fl. oz. Thus, rte of 1 to 4 qt of 5% formultion is too low to provide consistent effective disese control. In two other orchrds with copper-sensitive popultions of the pthogen, Regli (32 fl. oz. of 5%.i.)-Kocide 3000 or Regli-Quintec tretments reduced the disese from pproximtely 30% in the untreted control to pproximtely 8%. Regli-Kocide 3000 mixtures provided enefit over Regli lone when copper resistnce levels in the pthogen popultion re low or moderte. Regli cn e effectively used for wlnut light mngement under conditions of low disese pressure. 4. Among new tretments, tretments pplied to soil tht ffect stomtl opening of plnts (CoMo Clssic, RootZone) were effective under very low disese pressure (4.3% disese in the control). Soil pplictions with the systemic cquired resistnce (SAR) compound Actigrd tht were followed y folir Regli tretments significntly reduced disese incidence in the sme tril. In comprison of new non-copper nd iologicl tretments in study with 30% disese in the control, severl products including the iocontrols Actinovte nd Blossom Protect, s well s the experimentl AN-77V3 showed intermedite efficcy. Moleculr studies on pthogen diversity re ongoing nd demonstrte tht the pthogen hs limited genetic diversity within orchrds ut high diversity mong orchrds cross the stte. Popultion studies of cteril species in femle wlnut uds indicted tht t lest eight different species with yellow colonies co-exist with the pthogen. Disese levels in orchrds mesured in erly summer did not correlte well with ud popultions in the fll s determined with the use of selective isoltion medi nd Xnthomons-specific primers. A strtegy of using smll smple of 30 to 50 uds nd culturing cteril popultions on selective medi is not Cliforni Wlnut Bord 311 Wlnut Reserch Reports 2012

4 dvised t this time for predicting susequent spring disese levels nd mngement prctices. Evluting disese levels in orchrds in erly summer to identify high-risk orchrds nd using wether forecsts in the susequent spring (fter the pistillte-flower-emergence ppliction of ctericide) is the most effective wy to optimize dditionl tretment timings in mnging the disese. INTRODUCTION Wlnut light cused y the cterium Xnthomons roricol pv. juglndis (Xj) is continuous thret for Cliforni wlnut growers, especilly in centrl nd northern loctions where precipittion in the springtime is generlly higher. The incidence of disese vries widely etween yers, ut crop losses cn e very high s hppened t some loctions in The pthogen infects ctkins, femle lossoms, fruit, green shoots, leves, nd uds of English wlnut. Fruit infections occur soon fter flowering nd these infections ccount for most of the economic loss. Infections of other plnt prts contriute to continuous supply of inoculum. The extended period of host susceptiility nd the vilility of few effective tretments re the min chllenges in controlling wlnut light. The pthogen survives from one yer to the next in twig lesions, living nd ded uds, nd disesed fruit tht remin on the tree. Bsed on knowledge of environmentl requirements of the pthogen, nlysis of wetness period durtion nd temperture dt from field wether sttions, s well s ctul disese progression, we developed XnthoCst s model to predict infection periods for wlnut light nd clculte the risk of disese. The wesite currently provides the sic XnthoCst informtion with XnthoCst indices for wether sttions locted etween Red Bluff nd Dvis. The XnthoClcultor llows individul we-sed forecsting for different grower fields nd loctions. Thus XnthoCst system provides regionl forecsts nd promotes judicil use of pesticides with disese control similr to clendr-sed progrms. The UC-IPM wesite is under development. The min prolem is lck of lef wetness dt on CIMIS. Dt sets would need to e uploded nd then nlyzed. This is not just prolem for XnthoCst ut for mny models tht predict disese or insect outreks. Our reserch gol is to prepre XnthoCst for the next genertion of computerized systems tht push dt from utomted computer models using rel time-dt in finl form to end-users such s growers nd PCAs. This will eliminte the need for end users to go on-line, uplod dt, nd clculte indices on wesites (which is required for most predictive pest models). Insted, dt will e utomticlly sent to emil ddresses or cell phone pplictions of the end-users. Thus, our reserch pln is to convert XnthoCst to use dew point temperture, possily reltive humidity nd precipittion to identify lef wetness periods without ctully mesuring lef wetness. This chnge will offer considerle flexiility sed on dt tht re ville on CIMIS nd other dtses nd will llow the model to e utomted for future use. Our ctericide ppliction timing studies indicte tht erly femle flower emergence pplictions followed y tretments sed on XnthoCst provide the most consistent disese control. In our trils, ctkin ctericide tretment ws only needed when high precipittion occurred in n orchrd with history of high disese levels. We demonstrted tht single erdiction or inoculum reduction ud rek tretment is not sufficient to reduce the disese to cceptle levels in yers with moderte to high disese pressure. We lso showed tht cteril Cliforni Wlnut Bord 312 Wlnut Reserch Reports 2012

5 popultions cn rpidly (within 3 to 6 hours) nd exponentilly increse to very high levels on disesed fruit under fvorle environmentl conditions. Thus, inoculum levels re not considered in the XnthoCst model fter the initil tretment for light mngement. The model is continuously eing refined sed on new insights on the epidemiology of the disese. For exmple, ecuse higher tempertures inhiit growth of the pthogen, no pplictions re recommended when dily high tempertures exceed 27C regrdless of rinfll. For forecsting disese pressure nd the intensity of ctericide mngement progrms in the coming seson, others hve proposed tht the mgnitude of femle ud pthogen popultions s determined etween July nd lte winter cn e used. We re chllenging this concept sed on our epidemiologicl understnding of the disese where development of epidemics is more dependent on environmentl conditions thn on inoculum levels (see ove). In limited smpling in 2011, inoculum levels in 30- to 50-ud smples were not indictive of the mount of disese. Thus, Chndler nd other wlnut vrieties tht hd low to undetectle ud popultions in Fe. nd Mrch developed moderte levels of disese during the seson. In 2012, pthogen popultions were determined in uds collected in Novemer of 2012 from over thirty orchrds. Popultion sizes were compred to ctul disese incidences oserved in the 2012 seson nd will e compred to disese levels in the 2013 seson. For this ojective, we lso evluted the selectivity of different cteril isoltion medi nd determined which other cteri with yellow colony types interfered with enumertion of Xj colonies. Additionlly, in these studies we further evluted the specificity of our newly developed PCR primers for Xnthomons. This reserch is ongoing, ut preliminry dt will e presented in this report. Our ojective on the mngement of wlnut light since 1994 hs ssisted in the development of copper-ebdc (e.g., mne formulted s Mnex) tretments nd demonstrted the efficcy of zinc compounds nd zinc-mnex mixtures. We initited the rottion of copper- nd zinc-sed ctericides nd use of copper-zinc mixtures (e.g., Kocide 20/20, Nordox 30/30) to minimize the development of copper-resistnt popultions of the pthogen. In ddition, new reduced-copper products (e.g., Kocide 3000, Bdge) were shown to e highly effective. More recently, we showed tht products contining mncoze (Mnzte, Dithne, Penncoze, etc.) insted of mne re effective in mixtures with copper or the ntiiotic Ksumin. After the cncelltion of mne in the United Sttes, the EPA grnted emergency registrtions of Mnzte from 2010 to 2012 nd gin for Reserch on the effectiveness of Mnzte needs to e continued until full registrtion is otined. Copper-mncoze pplictions re currently the most effective tretment for wlnut light. Still, in 2012, the incidence of wlnut light t mny loctions ws very high (>50%), including in some orchrds where severl copper-mncoze tretments were done. This led to n investigtion s to the potentil cuse of this high disese level. We investigted if resistnce to mncoze could hve developed in some pthogen popultions. Additionlly, we lso considered other environmentl conditions, s well s chnges in mngement prctices tht could hve contriuted to this high disese incidence in some loctions. Over the yers, we evluted mny possile lterntives to copper-ebdc tretments. Among them, ksugmycin (Ksumin, Aryst Life Science) ws found to hve the gretest potentil in wlnut light mngement. This ntiiotic is used in griculture in other countries ut not in Cliforni Wlnut Bord 313 Wlnut Reserch Reports 2012

6 humn or niml medicine. Ksumin lone significntly reduced wlnut light incidence nd, under low rinfll, it ws highly effective. Under high rinfll (simulted with overhed irrigtion or nturlly occurring), mixtures of the ntiiotic with EBDC or copper resulted in n incresed efficcy nd outstnding performnce. Moreover, in most trils, Ksumin-EBDC or Ksumin-copper mixtures were similr to or higher in efficcy thn copper-ebdc mixtures. Currently, federl registrtion is trgeted for erly 2013 (i.e., Mrch 1) nd Cliforni registrtion possily lter tht yer. Idelly, we would like to hve some reserch-uthorized (e.g., RAs) demonstrtions this growing seson once federl registrtion hs een estlished on wlnut. Additionl reserch is lso needed on lterntive tretments such s nturl compounds (e.g., Regli, Citrox), iocontrols (e.g., Actinovte, Blossom Protect), enhncers of host resistnce (e. g., ProAlexin, Actigrd, ReZist, BioForge), nd nti-trnspirnts (e.g., Root-Zone, CoMo Clssic), s well s phosphonte pesticides reported to hve ctivity ginst cteril orgnisms including Xnthomons species. Some of these compounds demonstrted promising results efore. Actinovte ws registered for use in 2011 sed on our efforts nd is copper lterntive for orgnic growers. The nturl product Regli is mrketed s vile lterntive to copper-mncoze tretments. Our previous evlutions, however, hd indicted tht this tretment is only effective under low disese pressure. Thus, we re-evluted Regli gin in 2012 nd we lso used higher rtes thn the currently registered to find out if efficcy cn e incresed. Our overll gol for mngement of wlnut light is to ultimtely hve multiple tretments ville tht provide equl or improved efficcy to tht otined with trditionl copper-contining compounds. New tretments will lso need to e effective in orchrds with copper-sensitive nd copper-resistnt popultions of the wlnut light pthogen. This strtegy will result in lower usge nd decresed environmentl impct of ny one mteril nd lower potentil for selecting resistnt popultions of the pthogen. PROCEDURES Isoltion of Xj from disesed wlnut fruit nd determintion of in vitro sensitivities of isoltes of Xj ginst copper nd mncoze. Smples of lighted wlnut fruit were collected y frm dvisors, PCAs, nd chemicl industry representtives from 39 orchrd loctions in 10 counties. Tissue smples were suspended in sterile wter, liquots were streked onto yest-dextrose-clcium cronte (YDC) gr, nd single colonies were otined. Colonies were chrcterized y PCR using our new Xnthomons-specific primers developed from rdna sequence dt nd for their sensitivity to copper nd mncoze. Copper sensitivity ws determined y streking isoltes onto nutrient gr mended with 10 ppm or 50 ppm copper ion. Growth t 50-ppm copper fter 2 dys of incution t 25C ws considered copper-resistnt. Mncoze sensitivity ws tested using the spirl grdient dilution method. Bcteril suspensions were streked rdilly onto mended nutrient gr pltes nd incuted for 2 dys t 25C. Lowest concentrtions of mncoze where reduction of cteril growth ws oserved (LIC) nd concentrtions where growth ws inhiited y >95% MIC were determined y mesuring rdil growth nd then using computer progrm with n lgorithm sed on the moleculr weight of the test sustnce nd diffusion constnts. Evlution of lterntive ctericides for mngement of wlnut light in field studies. Trils were estlished in experimentl orchrds in Fresno Co. (Kerney Agriculturl Center - KAC) Cliforni Wlnut Bord 314 Wlnut Reserch Reports 2012

7 nd Solno Co. (UC Dvis), nd in commercil orchrds in Solno nd Sutter/Yu Co. In Fresno Co., tril ws conducted on cv. Chico. Simulted rin ws pplied in this orchrd for 7 h fter ech of the five-ctericide pplictions. Trils in the Solno Co. experimentl orchrds on cvs. Chico nd Hrtley were conducted under nturl rinfll conditions with ctericide pplictions sed on XnthoCst indices, nd four or five tretments were done. In the Solno Co. trils on cv. Tulre in commercil orchrd, the pthogen popultion ws determined to e prtilly (42.9%) resistnt to copper. Five pproximtely weekly pplictions were done. In the Sutter-Yu commercil orchrd tril with copper-resistnt popultion of Xj, five pplictions were done in 7- to 10-dy intervls. Tretment timings re indicted in Figs Copper compounds evluted included the microencpsulted copper hydroxide Kocide 3000 (DuPont Chemicl Co.), copper hydroxide (Chmp 30), pre-mixture of copper hydroxide/oxychloride (Bdge X2; Isgro, Inc.), nd sic copper sulfte (Cuprofix; United Phosphorus). Additionlly, the EBDC fungicide Mnzte ProStick 75DF, the phthlimide Cptn, the quinoline Quintec, nd the ntiiotic ksugmycin (Ksumin; Aryst Life Sciences) were used either lone or in selected mixtures. Nturl products/iocontrols evluted included Regli (n extrct of the plnt Reynoutri schlinensis; Mrrone Bio Innovtions); Actinovte ( fermenttion product of Streptomyces lydicus; Nturl Industries, Inc.); extrcts of citrus (Citrox nd ProAlexin; Citrox, Ltd); nd Blossom Protect (Aureosidium pullulns; Bio-Ferm). The nti-trnspirnts RootZone (GSI Horticulturl, Co.) nd CoMo Clssic (Stoller-USA), the SAR compound Actigrd (Syngent Crop Protection), the phosphonte K-Phite (Plnt Food Systems, Inc.), nd n cidified oxidizer (AN-77V2) were lso tested. Tretments were pplied using n ir-lst spryer t 100 gl/a, except for CoMo Clssic, RootZone, nd Actigrd tht were pplied to the soil in the Fresno Co. tril. For soil pplictions, 3.5 gl of solution ws pplied to the soil inside the drip line of ech tree t ech timing dte. Incidence of disese ws sed on the numer of infected fruit of fruit evluted for ech of four or five single-tree replictions. To determine disese incidence nd estimted yield (kernel weight) from eight trees from four rndomized five-tree replicted locks for ech tretment in the Sutter/Yu tril, counts of helthy nd disesed wlnuts t hrvest per stndrdized re were done. The ctul incidence of disesed nuts ws considered to e higher ecuse drop of disesed nuts during the seson ws not ccounted for in the disese ssessment t hrvest. High rinfll nd winds conducive for disese development were oserved in this orchrd. Dt were evluted using nlysis of vrince or generl liner model procedures nd lest significnt difference (LSD) men seprtion procedures of SAS 9.1. Quntifiction of Xj popultions in wlnut uds from commercil orchrds in the fll of 2012 nd correltion with disese levels in the previous seson, evlution of isoltion nd culture medi, nd specificity of our new Xnthomons-specific primers. Thirty to fifty femle flower uds were collected in Novemer of 2012 y PCAs in thirty-five orchrds tht hd disese levels from low (<5%), to moderte (5-20%), to high (>20% disesed nuts) in erly summer of Buds were split in hlf to e used for cteril isoltion nd future qpcr mplifictions. For cteril isoltion, 30 uds were used from ech loction. Bud hlves were cut into smll pieces, trnsferred into 1.7-ml microcentrifuge tues (3 uds/tue), 1 ml sterile distilled wter ws dded, nd tues were vigorously shken for 5 minutes. Superntnts were Cliforni Wlnut Bord 315 Wlnut Reserch Reports 2012

8 then diluted with sterile wter 1:100 nd spirled onto isoltion pltes using the exponentil deposition mode. For ll smples, the modified YDC medium ws used. Isoltion medi for suset of smples used were: Modified Tween Medium (Lindow; in Schd et l., 2001), Brillint Cresyl Blue Medium (Teviotdle et l.), Xn-D medium (Lee et l., 2009), YDC (yestdextrose-clcium cronte) gr, nd modified YDC (YDC mended with 1 mg fludioxonil, 33 mg cephlexin, nd 3.4 mg 5-fluoroucil/liter). Pltes were incuted t 25 C for 4 dys efore rting for cteril growth. A visul rting scle of colony forming units (e.g., cfu) ws utilized for colonies resemling Xj: 0 = no colonies, 1 = fewer thn 100 colonies, 2 = colonies, nd 3 = pltes contining more thn 200 colonies. These rtings were ctegorized s low (rting <1), moderte (1-1.7), or high (>1.7). Bcteril contminnts were rted using the sme scle. Single colonies of puttive Xj nd other yellow cteri were su-cultured onto YDC. A su-set of representtive isoltes ws lso cultured on ll five medi listed ove nd growth chrcteristics were noted. Representtives of puttive Xj colonies nd of yellow-pigmented contminnts were used in PCR mplifictions with our specific primers. Ten yellow-pigmented cteril colonies suspected not to e Xj were mplified with primers 63f nd 1387r tht trget the rdna gene region nd processed for DNA sequencing. Sequences were sumitted to BLAST similrity serch for species identifiction. Studies on genetic vriility within nd mong orchrd popultions in Cliforni. To evlute genetic vriility within nd mong orchrd popultions of the pthogen, DNA from selected strins tht were collected in 2012 ws mplified using rep-pcr primers BOX nd ERIC (Louws et l. 1994). PCR rections contined cteril cells nd stndrd regent mixtures. Amplifictions nd seprtion of mplicons using electrophoresis were done s descried previously. Ech strin ws mplified twice. Disese evlutions of wlnut genotypes in vriety lock t the Kerney AgCenter. Trees in the new wlnut vriety orchrd t Kerney Ag Center with new commercil cultivrs nd genotypes from the wlnut reeding progrm under Dr. Chuck Leslie were treted with six weekly 8-h simulted rin events using n impct sprinkler irrigtion system in the spring of Disese ws evluted in My nd June nd the numer of disesed fruit on ech tree ws counted. RESULTS AND DISCUSSION Wlnut light incidence in Cliforni in 2012 nd sensitivity of Xj popultions to copper nd mncoze. In 2012, incidence of wlnut light t mny loctions ws very high (>50%), including in some orchrds where severl copper-mnzte tretments were pplied. This led to n investigtion s to the potentil cuse of this high disese level. Becuse mncoze resistnce ws considered one possile cuse, isoltes of Xj were collected from 39 orchrd loctions in 10 counties. Isoltes were verified for their species identity using previously developed specific primers. Copper resistnce ws found in ll ut seven (including two experimentl orchrds t KAC in Fresno Co.) of the 39 orchrds tests in Butte, Monterey, Plcer, Sn Joquin, Solno, Stnislus, Sutter, Tehm, nd Yu Co. (Tle 1). Thus, copper-resistnce is still widespred throughout the wlnut production res of the stte. All 296 isoltes were found to e sensitive to mncoze. This informtion ws lso used to updte our dtse with modern identifiction Cliforni Wlnut Bord 316 Wlnut Reserch Reports 2012

9 methods nd to support the ongoing federl emergency registrtion of mncoze for wlnut in Cliforni. Bsed on this informtion, we concluded tht copper-mncoze tretments did not fil, ut tht environmentl conditions in 2011 nd 2012 were extremely fvorle for disese. High spordic rinfll hd occurred in lte My to lte June of 2011, fvoring the coloniztion of ud tissues for next yer s growth y the pthogen (Fig. 1). High rinfll lso occurred during wlnut loom nd erly fruit development in 2012 leding to spred of primry inoculum nd popultion growth. When rinfll ws ccompnied y high-wind conditions, cteril cells could hve een driven into fruit nd leves through the stomt. Thus, wind speed component is prt of epidemiologicl models for some cteril diseses. Torndo-like funnel clouds were reported t some loctions, including t one of our test sites in commercil orchrd. Mny of the initil symptoms oserved this yer developed t the lossom-end (i.e., end-light) of the fruit indicting tht infections occurred from primry inoculum in the uds, wheres some side infections (i.e., side-light) developed lter in the seson (e.g., My-June) s secondry infections from susequent wetness periods nd inoculum from primry infections (secondry inoculum). With high inoculum levels nd fvorle environments, protective tretments possily were not timed properly. Furthermore, reduced numer of pplictions proly could not protect the rpidly growing plnt tissues to provide effective ctericide residues to ll susceptile host tissues. This emphsizes the importnce of monitoring disese levels in the previous growing seson nd considering environmentl conditions of the previous nd current sesons in wlnut light epidemiology nd the potentil for disese development. Weekly pplictions were successful in mnging the disese in mny orchrds in northern Cliforni. Evlution of new formultions of copper, EBDC nd other fungicides, n ntiiotic, plnt growth regultors, iocontrols, nd nturl compounds for mnging wlnut light in field studies. Our ctericide efficcy trils on wlnut light in 2012 focused on the comprtive effectiveness of vrious copper products (Bdge X2, Kocide 3000, Cuprofix, Chmp 30) in mixtures with mncoze nd the ntiiotic Ksumin in mixtures with severl products including mncoze, copper, nd selected fungicides. Copper-mncoze tretments were effective in ll trils conducted, except t one commercil site where ll tretments, including grower-pplied tretments, were less effective in mnging the disese. In comprison of copper tretments in Solno Co. tril, Kocide 3000, Bdge X2, Cuprofix, nd Chmp 30 in mixtures with mncoze were sttisticlly similr in their effectiveness in mnging the disese. Bdge X2-mncoze provided the lowest incidence of disese (8.3% s compred to the control with 41.8%) (Fig. 2). Under high disese pressure (41.8% disese in the control), Ksumin hd n intermedite efficcy (Fig. 2). Under low disese pressure, however, tretments with Ksumin y itself were very effective in the Fresno Co. tril (Fig. 3). When Ksumin ws mixed with Mnzte or copper, the tretment ws sttisticlly similr in effectiveness s to copper-mnzte tretments (Figs. 2, 3, 5) in reducing the disese. Thus, over the yers, Ksumin mixed with copper or n EBDC (e.g., mne or mncoze) hs shown consistent high efficcy for controlling wlnut light. The federl registrtion dte is set for Mrch 2013 with Cliforni registrtion in erly Comintions of Ksumin with copper or mncoze in rottion with copper-mncoze re plnned s prt of the first integrted mngement strtegy of highly effective mterils ginst resistnt popultions of the pthogen. Cliforni Wlnut Bord 317 Wlnut Reserch Reports 2012

10 In the Fresno Co. tril, disese pressure ws very low nd only 4.3% of fruit in the untreted control developed light symptoms (Fig. 3). Most tretments significntly reduced disese incidence, including Bdge X2, Ksumin, Regli-Kocide 3000, Ksumin-Bdge X2, Ksumin- Mnzte, nd the phosphonte Ko-phite. This ltter mteril my induce host resistnce or lower the ph of the plnt sp nd decrese the susceptiility of the plnt to cteril pthogens tht prefer neutrl to lkline environments. Among new tretments, soil pplictions with RootZone or CoMo Clssic tht were used to induce stomtl closure to lock this cteril entry wy were effective under very low disese pressure (4.3% disese in the control) (Fig. 3). Soil pplictions with the plnt defense stimultor Actigrd tht were followed y folir Regli tretments significntly reduced disese incidence in the sme tril. RootZone showed very promising results in 2010, ut in 2011, oth RootZone nd CoMo Clssic hd little or no effect in reducing light. In these ltter trils, trees were very lrge nd older; wheres in the 2010 studies trees were smller nd younger. Thus, these tretments my hve to e limited to younger trees nd to southern wlnut production res of the stte where disese pressure is generlly lower. Still, this is revolutionry concept tht wrrnts further studies on stomtl function nd cisic cid production in treted plnts to confirm these nd other possile host responses. One tril t UC Dvis ws rte study with Regli. When pplied using 20%.i. experimentl formultion, only rtes of 64 fl. oz. (equivlent to 12.8 fl. oz..i.) or higher were effective (Fig. 4). These rtes, however, cused phytotoxicity. Leves initilly were chlorotic, reduced in size, nd lter ecme necrotic. Disese ws reduced from 19.3% in the control to 5.8 to 6.6%. This level of control is commercilly cceptle, however, the current mximum rte is 128 fl. oz. of the commercil 5%.i. formultion (equivlent to 6.4 fl. oz..i.) nd therefore too low to provide effective disese control y itself sed on the rte study (Fig. 4). The mximum lel rte is not cost-effective to growers if severl pplictions hve to e done during the seson. An dditionl rte study with rnge of 6.4 to 12.8 fl. oz. ctive ingredient of the commercil formultion is wrrnted. Regli-copper tretments were evluted in two orchrds with copper-sensitive popultions of the pthogen. In oth trils, Regli-Kocide 3000 reduced light incidence from pproximtely 30% in the control to pproximtely 8% (Fig. 5 no copper resistnce nd Fig % of pthogen popultion copper-resistnt). A mixture of Regli nd Quintec ws lso effective (Fig. 5). When copper ws dded to Regli s tnk mix, the performnce ws improved nd the tretment reduced disese to commercilly cceptle levels in one tril with significnt levels of copper resistnce this seson (Fig. 6). As previously documented, Regli cn e effectively used for wlnut light mngement under low disese pressure conditions. Regli-Kocide 3000 mixtures cn provide enefit over Regli y itself when copper resistnce levels in the pthogen popultion re low or moderte. Still, inconsistent performnce in multiple studies over severl yers prevents Regli (32 fl. oz./a)-copper (4 ls. Kocide 3000) mixtures to e considered replcement of copper-mncoze mixtures in conventionl orchrds ut the mixture is n lterntive for orgnic orchrds with copper resistnce. In tril in commercil orchrd in Solno Co., new non-copper nd iologicl tretments were compred. With 30% disese incidence in the control, severl products including the iocontrols Cliforni Wlnut Bord 318 Wlnut Reserch Reports 2012

11 Actinovte nd Blossom Protect nd the cidified oxidizer AN-77V3 provided intermedite efficcy (Fig. 6). Phytotoxicity s mrginl lef urn ws oserved using the 4-l nd 8-l rtes of AN-77V3. Furthermore, dditionl personl protection hd to e used during ppliction of this compound ecuse it ffects the respirtory trct. Wlnut kernel yields were determined t commercil site where five ctericide pplictions in 7- to 10-dy intervls were done etween nd Disese incidence nd yield were estimted t hrvest y counting totl nd helthy nuts per stndrdized re fter trees were shken nd nuts were on the ground under ech tree. Significnt differences etween tretments were shown for disese incidence nd estimted kernel weights per cre. Disese levels re lower thn the disese incidence for the seson ecuse disesed nut drop during nut development ws not included. Yields were lowest for the untreted control nd Bdge X2 (pplied y itself), intermedite for Regli-Bdge X2 nd Bdge-X2 Mnzte, nd highest for Ksumin-Mnzte (Fig. 7). In conclusion of the mngement studies, mong currently ville tretments, coppermncoze (EBDC) comintions re the most consistent in controlling wlnut light in Cliforni. Ksumin ws gin promising new tretment. Although modertely effective y itself, it performed etter in comintion with copper or mncoze. Ksugmycin (i.e., Ksumin) is pending US-EPA registrtion. As in previous yers, our reserch demonstrted tht pplictions of copper-mncoze t pistillte flower emergence followed y susequent sprys sed on temperture nd rinfll (s summrized in the XnthoCst model) is currently the most effective strtegy in mnging the disese. Potentilly in the future, ksugmycin-copper or - EBDC comintions in rottion with copper-ebdc tretments cn minimize the totl mount of ny ctericide eing used while mintining high levels of disese control nd providing resistnce mngement y lternting different modes of ction. Regli-copper comintions cn e effective under low to moderte disese levels nd for orgnic production of wlnuts where use of mncoze is not llowed. Neither Regli nor Ksumin cn e considered s potentil replcements of mncoze ecuse copper-mncoze mixtures hve twenty-yer history of proven consistent performnce for mnging wlnut light under low to highly fvorle conditions for disese. Evlution of isoltion nd culture medi for Xj; specificity of our new Xnthomons primers; nd quntifiction of Xj popultions in wlnut uds from commercil orchrds in the fll of 2012 nd correltion with disese levels in the 2012 seson. In studies to determine if ny of the five cteril culture medi used could esily differentite growth of Xj from tht of other cteri with yellow colonies, the Xn-D medium showed the highest selectivity (Tle 2). After two to three dys of growth, colonies of numerous strins of Xj tested were greenishglossy nd were surrounded y distinct luish-green milky hlo. This color chnge is due to chnge in ph of the growth medium from pproximtely ph 6.0 to 7.6. These strins of Xj lso ll yielded n mplifiction frgment of the expected size using our new Xnthomons-specific PCR primers. Some of the non-xj cteri, however, hd similr ppernce. Still, in contrst to the other four medi evluted, Xj could e differentited y these chrcteristics t higher frequency on the Xn-D medium. Cliforni Wlnut Bord 319 Wlnut Reserch Reports 2012

12 When used for recovering Xj in isoltions from femle wlnut uds, however, the chrcteristics of greenish-glossy colonies with luish-green, milky hlo on Xn-D medium did not prove stle when single colonies of Xj were growing mong multiple fungl nd cteril contminnts. Smll Xj colonies generlly did not exhiit the indictive color. Additionlly, some of the contminting microorgnisms lso ppered to ffect the ph of the medium, giving it lue color, so the luish-green, milky hlo of Xj could not e oserved. Low selectivity ws lso found for the Modified Tween Medium tht resulted in milky white hlo of mny non-xj cteri. The Brillint Cresyl Blue Medium lso did not prove to e highly selective, ecuse Xj colonies could not e esily differentited, nd dditionlly, growth ws very slow. The modified YDC medium ws rted s the most selective nd ws then used for ll ud isoltions (Tle 2). Fungl growth ws inhiited y fludioxonil nd growth of numerous other cteri y cephlexin nd 5-fluorourcil. Still, yellow colonies other thn Xj were commonly present. Representtive colonies were su-cultured on Xn-D to oserve colony chrcteristics such s colony color nd hlo nd were further tested for their identity y Xnthomons-specific primers using PCR or y sequence nlyses of the rdna region. Sequence nlysis of limited collection of ten yellow contminnts indicted tht strins elonged to the gener Arthrocter, Curtocterium, Enterocter, Frigoricterium, Mssili, Microcterium, Pntoe, nd Terrcter. In contrst to these isoltions from uds, isoltions from lighted wlnut fruit resulted in mostly pure colonies of Xj with few contminnts present. All cultures of Xj otined from disesed fruit in our survey of copper- nd mncoze sensitivity gve n mplifiction product of the predicted size using our new PCR primers. This is further support for the specificity of these primers for Xnthomons nd their usefulness in identifying unknown cultures. We hve evidence, however, tht these primers lso mplify very closely relted cteri such s X. roricol pv. pruni which cuses cteril spot of Prunus spp. In the identifiction of cteril species from wlnut this is considered not prolem ecuse it is unlikely to find species other thn Xj on this host. Unfortuntely, inocultion studies on wlnut trees re difficult to perform when numerous cteril strins hve to e evluted. Therefore, the use of culturl chrcteristics in comintion with PCR mplifiction is the most efficient wy to chrcterize cteril strins. Isoltion frequencies of Xj from femle wlnut uds from 35 orchrd loctions were ctegorized s low (rting <1), moderte (rting 1-1.7), or high (rting >1.7), nd were compred to disese levels oserved in erly summer (Tle 3). For 14 orchrds with low ud popultions, four orchrds hd moderte or high level of light, resulting in 28.6% error in predicting disese incidences from Xj ud popultion sizes. Disese rtings were ll moderte or high mong orchrds with moderte or high ud popultion levels. Still, 55.6% of orchrds with moderte levels of ud popultions hd high levels of disese nd 16.7% of orchrds with high ud popultions hd moderte levels of disese. These dt indicte tht correltions etween Xj ud popultion nd ctul disese levels re not cceptle. Any orchrd where the disese potentil is considered low sed on low ud popultions nd where susequently only miniml light mngement progrm is prcticed would e gret loss to the grower nd PCAs involved when the ctul disese level is high. Cliforni Wlnut Bord 320 Wlnut Reserch Reports 2012

13 Bud popultions will e evluted gin in the sme orchrds in erly spring of 2013 to determine the correltion etween popultion sizes in the spring nd disese levels in the coming seson. We elieve tht n lterntive nd simpler pproch to estimte the disese potentil is the ctul disese incidence in the previous seson. Determining incidence of disese in severl-hundred nut smple in June or July is inexpensive nd much esier nd more ccurte thn collecting uds, shipping, nd culturing of cteri from uds. Potentilly, qpcr pproch using Xnthomons-specific primers tht we re plnning to develop my still llow quntifiction of pthogen popultions in uds. At present, using 30- to 50-ud smples nd culturing cteril popultions using selective medi is not dvised for predicting susequent spring disese levels nd for plnning mngement prctices. Using ctul disese informtion from the pst seson to identify high-risk orchrds long with the forecsted environmentl conditions (e.g., tempertures nd wetness) during the spring (fter the pistillte-floweremergence ppliction of ctericide) re the most criticl prmeters to optimize timings of ctericide tretments in wlnut light mngement progrm. This is the sis of wlnut light mngement in Cliforni nd is summrized in the forecsting model XnthoCst. Studies on genetic vriility within nd mong orchrd popultions in Cliforni. Moleculr popultion studies continued to demonstrte tht multiple strins of the pthogen re present in Cliforni. In mny orchrds, one to two strins were found. In one orchrd from Monterey Co., however, mixed popultions of t lest three distinct strins were identified. Thus, we continue to demonstrte tht within specific loction only limited numer of strins exist lthough the pthogen is geneticlly diverse within the stte. Vrietl susceptiility. Disese ws evluted in our new vriety orchrd t Kerney Agriculturl Reserch nd Extension (KARE) Center, however, disese incidence ws extremely low. This is reltively new orchrd tht ws plnted two yers go. Thus, fvorle conditions for wlnut light development will e continued to e provided in the coming sesons y pplying overhed irrigtion to simulte rinfll. This ws our strtegy in the previous vriety orchrd where disese incresed from very low levels to high levels over three to four sesons using the sme strtegy. ACKNOWLEDGMENTS Specil thnks to the growers in Solno nd Yu/Sutter Co. who llowed us to conduct our reserch in their orchrds nd to the chemicl industry representtives who cooperted with us with during this reserch. REFERENCES 1. Adskveg, J. E, et l Annul Wlnut Reports Wlnut Mrketing Bord of Cliforni. Scrmento, CA. 2. Christofferson, C., McGrnhn, G., Mills, N., nd Leslie, C Annul Wlnut Reports Wlnut Mrketing Bord of Cliforni. Scrmento, CA. 3. Conover, R. A. nd Grenhold, N. R Mixtures of copper nd mne or mncoze for control of cteril spot of tomto (Xnthomons cmpestris pv. vesictori) nd their comptiility for control of fungus diseses. Proc. of Fl. Stte Hortic. Soc. 94: Cliforni Wlnut Bord 321 Wlnut Reserch Reports 2012

14 4. Lee, Y. -A., M. N. Schroth, M. Hendson, S. E. Lindow, X. -L. Wng, B. Olson, R. P. Buchner, nd B. Teviotdle Phytopthology 83: Lee, Y.-A., Sung, A.-N., Liu, T.-F., nd Lee, Y.-S Appl. Environm. Microiol. 75: Louws, F.J., Fulright, D.W., Tylor Stevens, C., nd de Bruijn, F.J Appl. Environ. Microiol. 60: Miller, P. W. nd W. B. Bollen Oregon Agric. Exp. Stn. Tech. Bull pp. 8. Schd, N.W., Jones, J.B., nd Chun, W Lortory Guide for Identifiction of Plnt Pthogenic Bcteri. Third Edition. APS Press, St. Pul, MN. 9. Scortichini, M, Mrchesi, U, nd Di Prospero, P J. Phytopthology 149: Teviotdle, B. L., M. N. Schroth, nd E. N. Mulren In: Wlnut Orchrd Mngement. Ed. y E. Rmos. Univ. of Clif. Coop. Ext. Pul Young, J.M., Prk, D.-C., Shermn, H.M., nd Frgier, E Syst. Appl. Microiol. 31: Cliforni Wlnut Bord 322 Wlnut Reserch Reports 2012

15 Tle 1. Survey for copper nd mnzte sensitivity in popultions of pv. juglndis from wlnut orchrds in Cliforni 2012 Xnthomons roricol Mncoze sensitivity Orchrd loction No. of Incidence of Cu LIC (mg/l) MIC (mg/l) (county) isoltes tested Resistnce (%)* Rnge Avg. Rnge Avg. 1 Butte Butte Butte Butte Butte Butte Butte Fresno Fresno Monterey sensitive sensitive 11 Plcer Sn Joquin Sn Joquin Sn Joquin Sn Joquin Sn Joquin Sn Joquin Sn Joquin Solno Stnislus Stnislus Stnislus Stnislus Sutter Sutter Sutter Sutter Sutter Tehm Tehm Tehm Tehm Tehm Tehm Tehm Tehm Tehm Yu undetermined totl 39 totl 296 Averge LIC 1.32 Averge MIC 2.13 * - Copper sensitivity ws tested using CuSO 4-mended nutrient gr pltes. Copper resistnt: growth t 50 mg/l Cu ion (200 mg/l CuSO 4) **- Mncoze sensitivity tested using the spirl grdient dilution plte method. LIC = lowest concentrtion of mncoze where reduction of cteril growth ws oserved; MIC = concentrtion of mncoze growth ws inhiited y >95%. ***- The two loctions in Fresno Co. were experimentl orchrds. Cliforni Wlnut Bord 323 Wlnut Reserch Reports 2012

16 Tle 2. Comprison of gr medi for culturing nd isoltion of Xnthomons roricol pv. juglndis Medium* Xj growth rte Xj colony color Chrcteristics of hlo Usefulness for identifiction Selectivity for isoltion Modified Tween Medium fst light yellow/yellow milky white low low Brillint Cresyl Blue Medium slow green milky low low Xn-D Medium fst green lue/green good low Yest Dextrose Cronte (YDC) fst yellow no hlo low low Modified YDC fst yellow fint white low *- See text for detils out medi. modertegood Cliforni Wlnut Bord 324 Wlnut Reserch Reports 2012

17 Tle 3. Comprison of Xj popultion sizes in wlnut uds in the fll of 2012 with ctul light rtings in erly summer t 35 orchrd sites * - Buds were collected in Novemer Isoltion pltes were rted fter 3 dys of incution t 25C sed on scle from 0 = no colonies to 3 = high density of Xj colonies. Numers re the verge of 10 isoltion pltes using 3 uds per plte. ** - Disese in the orchrd ws evluted in erly summer nd ws rted s low (<5% disese), moderte (5-20% disese, or high (>20% disese). ***- The error ws clculted s the pertentge of mismtches etween isoltion frequency nd ctul disese where isoltion frequency ws grouped in low (rting of <1), moderte (rting of 1-1.7), or high (rting of >1.7) recovery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liforni Wlnut Bord 325 Wlnut Reserch Reports 2012

18 Temperture (C), Precipittion (mm) Temperture (C), Precipittion (mm) Apr Fig. 1. Temperture nd precipittion summry for three loctions in northern Cliforni f or April-June in 2011 nd Apr Precip (mm) Dvis Avg Temp (C) 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun Precip (mm) Dvis Avg Temp (C) 1-Apr 8-Apr 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun Precip (mm) Durhm Avg Temp (C) 1-Apr 8-Apr 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun Precip (mm) Durhm Avg Temp (C) 1-Apr 8-Apr 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun Precip (mm) Avg Temp (C) Fir O ks Apr 8-Apr 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun 0 Precip (mm) Avg Temp (C) Fir O ks Apr 8-Apr 15-Apr 22-Apr 29-Apr 6-My 13-My 20-My 27-My 3-Jun 10-Jun 17-Jun 24-Jun Note: High rinfl in mid-my nd June of 2011contriuted to higher disese nd incresed inoculum nd susequent high disese with fvorle conditions (high rinfl nd higher tempertures) in April Fig. 2. E ffic c y of new c opper- nd non-c opper-s ed tretm ents for m nging wlnut light on c v. Tulre under nturl rinfll c onditions - S olno Co Tretment Rte Control --- Ksumin 8L + Cptn 80WP 100 ppm + 5 l Ph-D 6.2 oz Ksumin 8L 100 ppm Ksumin 2L + Quintec 100 ppm + 7 fl oz Ksumin 8L + Ph-D 100 ppm Quintec 7 fl oz Cptn 80WP 5 l Mnzte Prostick 75DF 2.4 l Ksumin 8L - Mnzte ProStick 75DF 100 ppm l Cuprofix + Mnzte ProStick 75DF 5 l l Bdge X2 4 l Chmp 30 + Mnzte ProStick75DF 4 l l Kocide Mnzte ProStick 75DF 4 l l Bdge X2 + Mnzte ProStick 75DF 4 l l Disese incidence (%) g c cd cde cde def def def defg defg efg efg fg Prtil c opper res is tnc e 5 pplictions were mde on 4-19, 4-24,5-2,5-9, nd Disese ws evluted in lte My Incidence ofdisese is sed on 100 fruitfor ech of4 single-tree replictions. Cliforni Wlnut Bord 326 Wlnut Reserch Reports 2012

19 Fig. 3. Efficcy of new non-copper-sed nd iologicl tretments for mnging wlnut light on cv. Chico under nturl nd simulted rinfll conditions - Fresno Co No copper resistnce Tretment Rte Disese incidence (%) Control --- Citrox BC + ProAlexin 0.1% - 0.1% Mnzte Prostick 75DF 2.4 l c Ksumin 2L - Mnzte 75DF 100 ppm l c Bdge X2 + Mnzte ProStick 75DF 4 l l c Ksumin 2L + Bdge X2 100 ppm - 4 l c Actigrd soil + Regli folir 1 oz + 32 fl oz c CoMo Clssic soil 16 fl oz c Ko-phite 96 fl oz c RootZone soil 500 fl oz c Ksumin 2L 100 ppm c Bdge X2 4 l c Regli 5% + Kocide fl oz - 4 l c pplictions were mde on 4-18, 4-24, 5-2, 5-18, nd One dy fter ech ppliction simulted rin ws pplied for 7 h using overhed sprinklers. For soil pplictions, 3.5 gl solution ws pplied to the soil inside the drip line of ech tree t ech timing dte. Disese ws evluted in erly June. Incidence of disese is sed on 100 fruit for ech of 4 single-tree replictions. Fig. 4. Efficcy of selected rtes of Regli for mnging wlnut light on cv. Chico under nturl rinfll conditions - Solno Co No copper resistnce Tretment Rte Control ---- Regli MBI fl oz Regli MBI fl oz Regli MBI fl oz Regli MBI fl oz Disese incidence (%) Phytotoxicity rting pplictions were mde on 4-6, 4-16, 4-25, 5-3, nd with 20% ctive ingredient formultion of Regli (the commercil formultion contins 5%.i.). Disese ws evluted in lte My. Incidence of disese is sed on 100 fruit for ech of 4 single-tree replictions. Rting of phytotoxicity ws sed on scle from 0 (= no symptoms) to 4 (= >75% of the leves of reduced size nd with chlorosis or necrosis. Fig. 5. Efficcy of selected tretments for mnging wlnut light on cv. Hrtley under nturl rinfll conditions - Solno Co No copper resistnce Tretment Rte Disese incidence (%) Control ---- Ksumin 2L + Mnzte ProStick 100 ppm l Regli 5% + Quintec 32 fl oz + 7 fl oz Kocide Mnzte ProStick 4 l l 4 pplictions were mde on 4-23, 5-1, 5-8, nd Disese ws evluted in lte My. Incidence of disese is sed on 100 fruit for ech of 4 single-tree replictions. Regli 5% + Kocide fl oz + 4 l Cliforni Wlnut Bord 327 Wlnut Reserch Reports 2012

20 Fig. 6. Efficcy of new non-copper-sed nd iologicl tretments for mnging wlnut light on cv. Tulre under nturl rinfll conditions - Solno Co Prtil copper resistnce Tretment Rte Disese incidence (%) Phytotoxicity rting Control --- AN-77V2 2 l AN-77V2 8 l Actinovte + NuFilm 12 oz + 8 fl oz AN-77V5 + Cheltor + Kocide l l l Blossom Protect + Buffer 1.34 ls ls AN-77V5 + Cheltor 4 l l Regli 5% + Kocide fl oz + 4 l AN-77V2 4 l pplictions were mde on 4-19, 4-24, 5-2, 5-9, nd Disese ws evluted in lte My. Incidence of disese is sed on 100 fruit for ech of 4 single-tree replictions. Phytotoxicity ws rted using scle from 0 (= no symptoms) to 3 (= severe mrginl lef urn). Fig. 7. Disese incidence nd wlnut yields in commercil cv. Ashley orchrd in Yu/Sutter Co. using selected wlnut light mngement progrms Copper resistnce Tretment Rte Control --- Bdge X2 Regli 5% + Bdge X2 Bdge X2 + Mnzte ProStick Ksumin 2 L + Mnzte ProStick 4 l 32 fl oz + 4 l 4 l l 64 fl oz l Disese incidence (%) Five pplictions (7-10 dys prt) were done etween 4-5 nd Counts of helthy nd disesed wlnuts t hrvest per stndrdized re were used to determine disese incidence nd estimted yield (kernel weight) from eight tree replictes for ech tretment. Totl disesed nuts ws considered to e higher ecuse disesed nut drop during the seson ws not ccounted for in the disese levels t hrvest. High rinfll nd winds conducive for disese development were oserved in this orchrd. Estimted yield (l/a) Fig. 8. Amplifiction of cteri with yellow-pigmented colonies otined from wlnut uds using Xnthomons-specific PCR primers Amplifictions were done using cteril cells directly in PCR rection mixtures. Of the 24 strins evluted in this gel, 12 resulted in n mplifiction frgment. These cultures showed typicl chrcteristics on Xn-D growth medium (see text). The first lne of the gel is 100-p size stndrd. Cliforni Wlnut Bord 328 Wlnut Reserch Reports 2012